PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4

Abstract

Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.

Figure 1.

PP32 and SET/TAF-Iβ are components of the Complex Ib. (A) Scheme illustrating the purification procedure for obtaining Complex Ib from HeLa S100 extracts. (B) Western blots of fractions derived from the last DEAE-5PW column, as indicated. The corresponding salt concentration of the fractions and the elution of Complex Ib and Complex II are specified at the bottom. Complex Ib elutes in fractions 21–22 and Complex II in fractions 40–42. An additional uncharacterized histone H3 and H4 peak is observed from fraction 48. (C) Left, silver staining of the enriched Complex Ib, corresponding to the pool of fractions 19–23 derived from the last DEAE-5PW column. Right, mass spectrometry (MS) data of the enriched Complex Ib, indicating the number of peptides identified for each protein and the coverage. (D) Western blot of the enriched Complex Ib, corresponding to the pool of fractions 19–23 derived from the last DEAE-5PW column.

Figure 2.

PP32 and SET/TAF-Iβ associates with newly synthesized histone H4. (A) Scheme illustrating the procedure utilized to label and isolate newly synthesized proteins. (B) Western blot analysis of 1% of the input material (left), purified Flag-immunoprecipitated proteins (middle), and Streptavidin–agarose pulled-down proteins derived from the Flag-immunoprecipitated material (right), as indicated. Top panels were developed with Streptavidin-HRP to visualize AHA-labeled proteins conjugated to biotin AHA–biotin. Bottom panels were developed as indicated. (C) Western blot analysis of 1% of the input material, purified Flag-immunoprecipitated proteins and Streptavidin–agarose pulled-down proteins derived from the Flag-immunoprecipitated material, with extracts in which the CLICK-IT procedure was performed with DMSO instead of AHA. Top panel was developed with Streptavidin–HRP to visualize AHA-labeled proteins conjugated to biotin AHA–biotin. Bottom panels were developed as indicated.

Figure 3.

PP32 and SET/TAF-Iβ proteins regulate newly synthesized H4 acetylation levels in vivo. Western blots of 10 and 30 μg of S100 extracts derived from either siControl and siPP32 (A) or siSET/TAF-Iβ (B) treated HeLa cells.

Figure 4.

PP32 and SET/TAF-Iβ proteins block HAT1 mediated H4 acetylation in vitro. Acetylation assay performed using 0.128 nmol of recombinant histone H4 and purified HAT1 in the presence or absence of increasing amounts of recombinant PP32 (A) and recombinant SET/TAF-Iβ (B), followed by H4K12ac Dot-blot detection. Top: graph of the remaining HAT1 activity. 100% activity correspond to the HAT1 mediated H4 acetylation in the absence of PP32 and SET/TAF-Iβ. Bottom: a representative H4K12ac dot blot HAT1 assay. FT corresponds to the flow through material of the recombinant SET/TAF-Iβ Ni⁺²-beads purification. (C) Top: Coomassie blue stained gel of the different recombinant proteins used in the acetylation assay. Bottom: acetylation assay performed as in (A), pre-incubating H4 as indicated. (D) Acetylation assay performed using recombinant histone H4 and purified HAT1 in the presence of 8 pmol of recombinant PP32, 12 pmol of recombinant SET/TAF-Iβ, and a mix of 8 pmol of recombinant PP32 and 12 pmol of recombinant SET/TAF-Iβ (Mix 1×), and 4 pmol of recombinant PP32 and 6 pmol of recombinant SET/TAF-Iβ (Mix 0.5×).

Figure 5.

PP32 knock-down affects the maturation of newly synthesized histone H4. (A) Western blot of HA-Hsp90 pull-down assay from cytosolic extracts derived from siControl and siHsp90 treated HeLa cells. (B) Graph of the tryptophan fluorescence spectroscopy of Hsp90 upon titrating full length histone H4 (left), or unmodified or acetylated amino acids 1–20 of histone H4 (right). ΔF is the difference between initial fluorescence (F₀) and the fluorescence after ligand addition (F_Q). K_D is the dissociation constant of the ligand with Hsp90. (C) Left, Western blot analysis of thermal stability assay using HeLa S100 extracts. 20 μg of S100 extracts were heated for 20 min at the indicated temperatures and centrifuged at 10 000 × g for 5 min. Supernatant and pellet were analyzed by western blot. Right, graph of the percentage of either total or acetylated histone H4 found on the pellet, taken as 100% of the sum of the supernatant and pellet at each temperature. Standard deviations were taken from three independent experiments. *P < 0.05, Student′s t-test. (D) Thermal stability of S100 extracts derived from siControl and siPP32 HeLa cells, as described in (C). (E) Thermal stability of S100 extracts derived from siControl and siPP32 HeLa cells, as described in (C), in the absence or presence of recombinant PP32.

Figure 6.

PP32 knock-down affects cell cycle progression. (A) Cell cycle profiles of HeLa cells treated with siControl, siPP32, and siSET/TAF-Iβ. (B) Proposed model for the regulation of newly synthesized histone H4 acetylation. After synthesis, unacetylated histone H4 interacts with the heat shock proteins Hsp90 and Hsp70 and two members of the INHAT complex: PP32 and SET/TAF-Iβ. The binding of PP32 and SET/TAF-Iβ protects histone H4 from premature acetylation by HAT1, as well as for non-specific acetylation mediated by other HATs. When PP32 protein levels are diminished, histone H4 is acetylated at early steps of the maturation cascade, and it no longer can interact with Hsp90 affecting its stability and maturation.